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Keywords:

  • Phlebotomus papatasi;
  • P. tobbi;
  • P. sergenti;
  • genetic structure;
  • nucleotide diversity;
  • population extension;
  • leishmaniasis;
  • Turkey

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

The object of this study was to determine the genetic structures of three vector species, Phlebotomus tobbi, Phlebotomus papatasi, and Phlebotomus sergenti, in the Cukurova Region of Turkey, an endemic focus of cutaneous leishmaniasis. The genetic diversity indices, neutrality tests and hierarchical analysis of molecular variance (AMOVA) were performed using partial sequences of ITS2 and cytochrome b gene regions. In all species, within population genetic variation was higher than between population variation for ITS2 gene region. Fst values were low and non-significant for P. sergenti, and were higher for P. papatasi and P. tobbi indicating a weak structuring between populations. AMOVA tests suggest any substantial isolation between populations within species. AMOVA analysis of cyt b gene region revealed significant genetic structuring between populations for P. papatasi and P. sergenti. Fst values were relatively high and significant for these species indicating a certain degree of isolation between populations. However, in P. tobbi, any significant population genetic structuring was detected. Tajima's D and Fu's Fs values were negative and significant in all three species might be indicating a demographic expansion.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

Leishmaniasis is transmitted by several different species of phlebotomine sand fly vectors (Volf et al. 2002). The disease is endemic in all the countries of the Mediterranean basin and its epidemiology is rapidly evolving (Schönian et al. 2008). As a Mediterranean country, Turkey represents a crossroad between the three continents of Africa, Europe and Asia, with ecological and climatic features that are important in the epidemiology of leishmaniasis. Two clinical types of leishmaniasis exist in Turkey. Human cutaneous leishmaniasis (CL) caused by Leishmania tropicaWright, 1903 (Alptekin et al. 1999, Akman et al. 2000, Volf et al. 2002) and L. infantum Nicolle 1908 (Serin et al. 2005, Svobodova et al. 2009), which is highly endemic in south and southeast Anatolia, and human visceral leishmaniasis (VL), caused by L. infantum, which is endemic along the Aegean and Mediterranean costs and occurs sporadically in other regions (Ozbel et al. 1995, Ok et al. 2002, Volf et al. 2002, Yaman and Ozbel 2004).

The Cukurova Basin has become a second endemic focus of CL within Turkey. Because it is an ecotone between western and eastern Anatolia, is one of the crossroads between Asia and Europe in terms of animal and plant dispersion, and almost 70% of the region is covered by agricultural area, the plain is appealing to a large human population. Accordingly, there is a significant increase of CL cases over the past 10 years. Most of the species found in this region are known vectors of CL (Simsek et al. 2007, Svobodova et al. 2009). According to recent studies, the etiological agent of CL in the region was identified as L. infantum, and Phlebotomus (Larroussius) tobbi Adler, Theodor et Lourie 1930 was shown to be the vector of CL in the Cukurova focus (Svobodova et al. 2009).

Populations are usually found in nature as separated subunits as a result of ecological, behavioral, or genetic diversification. These subpopulations may exhibit different phenotypic and genotypic features from each depending on such factors as gene flow, geographic distance and diversifying selection (Hendrick 2005). Strong genetic differentiation between phlebotomine sand fly populations are expected since they deposit their larvae into the soil and are weak fliers as adults, making them highly habitat specific (Depaquit et al. 2002). This genetic differential and subsequent adaptation to the current environment can cause differences in vectorial capacities of sand flies. Previous studies showed that P. tobbi is prevalent throughout Turkey and constitutes the most abundant species in the Cukurova region (Simsek et al. 2007, Erisoz Kasap et al. 2009). Therefore, it is necessary to determine the genetic structures of P. tobbi populations in order to improve our knowledge of leishmaniasis transmission in this area. Together with P. tobbi, the genetic structures of P. (Paraphlebotomus) sergenti Parrot 1917 and P. (Phlebotomus) papatasi Scopoli 1786 populations were examined because the former is the proven vector of L. tropica in the southeastern part of the Turkey (Alptekin et al. 1999, Akman et al. 2000, Volf et al. 2002) while the latter is another vector of L. major (Killick-Kendrick 1990, Parvizi et al. 2005), which was found in several places (Akman et al. 2000) and is largely distributed all over the country.

The aim of this study was to determine the genetic structure of P. tobbi, P. papatasi and P. sergenti populations within the Cukurova region. Determining the genetic structures of these populations will increase our knowledge of leishmaniasis transmission within this area. Population genetic structure was assessed using two genes; one nuclear, the internal transcribed spacer 2 (ITS2) gene region, and one mitochondrion, cytochrome b (cyt b) gene region. These two gene regions are known for their relative high mutation rates making them highly useful for studies at the intra population level (Esseghir et al. 1997, Mukabayire et al. 1999, Depaquit et al. 2000, 2002, Di Muccio et al. 2000, Yahia et al. 2004, Moin-Vaziri et al. 2007, Hamarsheh et al. 2007).

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

Study area

The Cukurova Basin is approximately 28,000 km2 in area surrounded by the West Taurus Mountains on the west, Taurus Mountains on the north, Amanos Mountains on the east, and the Mediterranean Sea on the south. Seyhan (on the west) and Ceyhan (on the east) rivers nourish the plain. The study was carried out at Tarsus village, Adana and Osmaniye provinces, and their environment (Figure 1, Table 1).

image

Figure 1. Map of the study area showing the main sites sampled, the main geographical barriers between the sites, cities. Triangles indicate the sampling sites.

Download figure to PowerPoint

Table 1.  Sampling stations in the study area.
VillagesCoordinatesAltitudeVillagesCoordinatesAltitude
 NorthEast  NorthEast 
Kulak (KUL)36 47 37.734 52 01.81 mDöşeme (DOS)37 25 28.135 51 59.2175 m
Dedepınarı (DDP)36 55 36.635 28 56.44 mCamili (CAM)37 20 19.535 36 38.5181 m
Adalı (ADA)36 38 04.835 32 33.07 mTepeçaylak (TCK)37 03 33.635 03 34.7188 m
Yemişli (YEM)36 39 18.635 21 31.09 mTepecikören (TEP)37 21 50.835 37 38.9189 m
Eğriağaç (ERA)36 46 40.835 26 50.49 mTehçi37 09 14.836 20 01.9192 m
Ziyalı (ZIY)36 49 35.435 34 22.418 mKarakütük (KKK)37 24 40.136 07 15.8217 m
Doğankent (DK)36 50 51.935 20 19.020 mSarımazı (SMZ)36 58 21.535 58 32.4222 m
Yenice (YEN)36 58 39.235 02 57.442 mDutlupınar (DUT)37 01 39.736 01 03.7222 m
Hamamköy (HAM)37 19 08.735 49 02.845 mOtluk (OT)37 18 05.235 31 05.3237 m
Narlık (NAR)36 55 03.635 51 14.348 mGökbüket (GOK)37 07 39.135 32 48.6244 m
İsalı (ISA)36 55 11.935 43 04.962 mAydın (AY)37 24 35.635 35 43.8278 m
Bucak (BUC)37 26 54.435 54 19.566 mZerdali (ZER)37 24 18.235 37 51.2292 m
Tumlu (TUM)37 08 49.935 42 26.366 mSofular (SOF)37 22 53.936 14 20.0316 m
Küçük Tüysüz (KTUY)37 02 57.036 05 31.478 mKızyusuflu (KIZ)37 19 54.836 12 36.7373 m
Baklalı (BAK)37 02 11.235 38 16.6124 mAkçakoyunlu (AKC)37 11 15.436 25 13.3373 m
Koyunevi (KOY)37 17 21.335 39 23.6146 mGedikli (GED)37 30 10.135 51 40.5381 m

The plain has a temperate climate with long summers and short and rainy winters. This climate enables the cultivation of three or more agricultural products per year. It is also preferable by sand flies and especially the warm season coincides with their reproductive cycles (Lewis 1982, Alptekin et al. 1999, Volf et al. 2002). Most of the area is fertile (mollisol and alluvial soil) and used for agricultural activities but Pinus and Abies forests are also cultivated. The villages are usually surrounded by citrus orchards and cotton fields, most commonly in the eastern part. Residents live in single-family houses built from briquette, adobe, stone and cement, surrounded by gardens with henhouses and sheep or cattle sheds.

Sand fly collection and identification

Seventy-one of the thousands of specimens collected from 32 villages of the study area were selected for determining the genetic structures of the populations (Table 2). Indoor and outdoor sand fly collections used CDC light traps (John W. Hock, U.S.A.), sticky papers and mouth aspirators (Alexander 2000). Specimens collected from each trap were stored in alcohol for morphological identification. Identifications were based on the morphology of male genitalia and female spermathecae and pharynges using the keys of Theodor (1948), Lewis (1982), Killick-Kendrick, (1991). Detailed information on the distribution, abundance and seasonal dynamics of species within the study area and within villages are given in Belen and Alten (2011).

Table 2.  Haplotype distributions of ITS2 and cyt b gene regions for the three species. H: ITS2 haplotypes; h: cyt b haplotypes. Frequencies of haplotypes are in parentheses.
VillagesAltitudeP. papatasiP. sergentiP. tobbi
  ITS2cyt bITS2cyt bITS2cyt b
Kulak (KUL)1 m h3    
Dedepınarı (DDP)4 mH2, H3h1, h3    
Adalı (ADA)7 m    H7h7
Yemişli (YEM)9 m    H1, H2, H3h1, h2, h3,h4
Eğriağaç (ERA)9 m    H6h6
Ziyalı (ZIY)18 mH3     
Doğankent (DK)20 mH4(4), H5, H6h3(6)    
Yenice (YEN)42 mH1h3    
Hamamköy (HAM)45 m     h14, h15
Narlık (NAR)48 m    H5h5(2)
İsalı (ISA)62 m    H5h8
Bucak (BUC)66 m     h13
Tumlu (TUM)66 m    H10h11
Küçük Tüysüz (KTUY)78 m     h5
Baklalı (BAK)124 m    H13h5
Koyunevi (KOY)146 m h3    
Döşeme (DOS)175 m     h17
Camili (CAM)181 mH3, H4h4 h5H9h9, h10
Tepeçaylak (TCK)188 mH4h1, h2    
Tepecikören (TEP)189 m h3, h6H2, H8h5 h2(2), h12
Tehçi192 m    H11h16
Karakütük (KKK)217 m     h2
Sarımazı (SMZ)222 m  H1, H2h1, h2  
Dutlupınar (DUT)222 m  H3, H4h1, h2  
Otluk (OT)237 mH6h5    
Gökbüket (GOK)244 m    H8h2
Aydın (AY)278 mH4h7H3h2(2)H12h2
Zerdali (ZER)292 m  H3h7 h18
Sofular (SOF)316 m   h6H10h5
Kızyusuflu (KIZ)373 m  H3, H5, H6, H7h6(3)H4, H5h2, h5
Akçakoyunlu (AKC)373 m  H3, H4h2  
Gedikli (GED)381 m  H8h3, h4H14h13
Total number of haplotypes 67871418
Total number of specimens 152014161731

DNA extraction, amplification and sequencing

DNA was isolated from thoraces following the procedures for the Qiagen DNeasy Blood and Tissue Isolation kit. The samples were dried before DNA extraction. PCR was performed in a 50 μl volume using 1.5 μl extracted DNA solution and 100 pmol of each of the two primers for ITS2. Primer sequences are: C1a: 5’–CCT GGT TAG TTT CTT TTC CTC CGC T-3’ and JTS3: 5’–CGC AGC TAA CTG TGT GAA ATC-3’ (Depaquit at al. 2000). The PCR mix contained 42.25 μl distillated water, 5 μl 10Xbuffer, 0.15 μl 25 mM dNTP, 1 μl 1.5 mM MgCl2 and 0.25 μl (1.25 unit) Taq polymerase (Bioron). Initial denaturation at 93° C for 1 min was followed by 35 cycles of denaturation at 94° C for 45 s, annealing at 56° C for 45 s, extension at 72° C for 45 s, and a final elongation time of 2 min at 72° C.

Similarly for the cyt b gene region, PCR was performed in a 50 μl volume using 1.5 μl extracted DNA solution and 2 μl (10 pmol=1/10 diluted) of each of the two primers were added. Primer sequences for cyt b are: CB1-SE: 5’-TAT GTA CTA CCC TGA GGA CAA ATA TC-3’ and CB-R06: 5’-TAT CTA ATG GTT TCA AAA CAA TTG C-3’ (Parvizi and Ready 2006). PCR mix was prepared using 35.75 μl distillated water, 5 μl 10Xbuffer, 3 μl 2.5 mM dNTP, 2 μl 1.5 mM MgCl2 and 0.25 μl (1.25 unit) Taq polymerase (Bioron). Initial denaturation at 94° C for 2 min was fallowed by 10 cycles of denaturation at 94° C for 30 s, annealing at 40° C for 30 s and extension at 72° C for 30 s. These cycles were followed by another 25 cycles of 94° C for 30 s, annealing at 48° C for 30 s and extension at 72° C for 30 s with a final elongation time 3 min at 72° C. Amplicons were analyzed by electrophoresis in 80 ml, 1.5% agarose gel containing 10 μl etidium bromide.

All PCR products were purified with the Invisiorb PCR Purification kit before sequencing. Purification products were both direction sequenced directly with the ABI Big Dye Terminator Cycle Sequencing kit with the primers used for DNA amplification reactions.

Sequence analysis

Sequences were aligned and edited using Clustal W2 software. Nucleotide diversity between and within populations were estimated using the Φ (Watterson, 1975) and π (Tajima, 1983) statistics. We also calculated neutrality indices Tajima's D and Fu's Fs using the mtDNA cyt b gene region in order to determine whether there was any deviation from the assumption of neutrality. Tajima's D and Fu's Fs indices were not calculated for the ITS2 gene region since multi-copy genes violate the assumptions of the tests. A molecular analysis of variance (AMOVA) was conducted in order to determine the genetic structure of populations. (Cockerham 1969, 1973, Weir and Cockerham 1984). Statistical significance of Fst values was determined using a non-parametric permutation approach (Excoffier et al. 1992). All analyses were performed using Arlequin v.3.1.1 (Excoffier et al. 2005) software.

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

Analysis of ITS2 sequences

Summary of genetic diversity indices, neutrality tests and results of hierarchical analysis of molecular variance (AMOVA) for all species using ITS2 gene sequences is shown in Table 3.

Table 3.  Summary of molecular results for ITS2 and cyt b gene regions of the three species.
SpeciesP. papatasiP. sergentiP. tobbi
Gene regionITS2cyt bITS2cyt bITS2cyt b
Number of individuals152014161731
Fragment length (bp)490639480625400653
A-T ratio (%)71,3370,2371,2769,8468,1573,85
G-C ratio (%)28. 6729,7728,7330,1631,8526,15
Number of haplotypes77871418
Haplotype diversity0.781±0.1020.516±0.1320.890±0.0600.850±0.0600.926±0.0580.905±0.038
Number of polymorphic sites107662268
Substitutions105661768
Transition54541346
Transversion5112423
Nucleotide diversityHighLowLowLowHighHigh
θS3.075±1.4371.409±0.7581.887±0.9961.808±0.9415.028±2.09317.021±5.518
θπ2.057±0.1020.936±0.7491.725±1.1991.441±1.0364.044±2.3759.815±5.137
Source of variation      
Among populations (%)31,9567,06–12,4666,6740,2717,75
Within populations (%)68,0532,94112,4633,3359,7382,25
Fst0.320; p=0.0140.670; p=0.002–0.126; p=0.1050.666; p=0.0000.402; p=0.0080.177; p=0.000
IsolationLowHighNot significantHighLowLow
Neutrality analyses      
Tajima – D–1,460–0,694–1,590
Fu FS–3,242*–26,060*–24,550*
ExpansionSignificantSignificantSignificant

The total length of sequences after alignment and pruning were 490, 480 and 400 bp for P. papatasi, P. sergenti and P. tobbi respectively. Sequences were A-T rich in all species (71.33% for P. papatasi; 71.27% for P. sergenti; 68.15% for P. tobbi). Appendix 1 contains the unique haplotypes obtained from ITS2 sequences for all species.

Within P. papatasi, we determined seven unique haplotypes containing 10 polymorphic sites with no indels. Total number of substitutions was 10 (5 transitions and 5 transversions). Nucleotide diversity was generally low (θs=3.075 ± 1.437; θπ=2.057 ± 1.368) however total haplotype diversity was high 0.781 ± 0.102. The distribution of P. papatasi haplotypes within the Cukurova region is shown in Figure 1A. Haplotype 5 (H5) was the most widespread haplotype fallowed by H3 while H2 and H6 were only found in one village.

Within P. sergenti we determined eight haplotypes containing 6 variable sites with no indels. Total number of substitutions was 6 with a transition-transversion ratio of 5:1. Similar to P. papatasi, nucleotide diversity was low (θs=1.887 ± 0.996; θπ=1.725 ± 1.199). Haplotype distributions are shown in Figure 1C. H3 was the most common haplotype while other haplotypes were mostly unique to one sampling location. Haplotype diversity was highest in the Kızyusuflu (KIZ) village (4 haplotypes).

Within P. tobbi we determined 14 unique haplotypes containing 22 polymorphic sites with five indels. A total of 17 substitutions was observed with a transition to transversion ratio of 13:4. Nucleotide diversity was relatively high (θs=5.028 ± 2.093; θπ=4.044 ± 2.375) and haplotype diversity was 0.926 ± 0.058. The distribution of P. tobbi haplotypes is given in Figure 1E, with nearly all haplotypes unique to sampling locations. The most widespread haplotype was H5 that was sampled from three locations.

Separate AMOVA analyses of P. papatasi, P. sergenti and P. tobbi populations, revealed no genetic structuring between populations within the Cukurova region (Table 3). In all species, within population genetic variation was higher than between population variation and variance components for between population variation was non significant (P. papatasi, 31.95%, Va=0.347, p=0.122; P. sergenti, –12.46%, Va=-0.106; p=0.653; P. tobbi, 40.27%, Va=0.824, p=0.073). In P. sergenti Fst values were extremely low and non-significant (Fst =–0.126, P = 0.105). Fst values for P. papatasi and P. tobbi were higher indicating a weak structuring between populations (FstP. papatasi=0.320; p=0.014; Fst P. tobbi=0.402; p=0.008). Overall AMOVA tests conducted separately for all three species suggests that there is no substantial isolation between populations within species.

Analysis of cyt b sequences

Summary of genetic diversity indices, neutrality tests and results of hierarchical analysis of molecular variance (AMOVA) for all species using cyt b gene sequences is shown in Table 3.

The total length of sequences after alignment and pruning were 639, 625 and 653 bp for P. papatasi, P. sergenti and P. tobbi, respectively. Similar to ITS2, the sequenced region was A-T rich for all species (70.23% for P. papatasi; 69.84% for P. sergenti; 73.85% for P. tobbi). Appendix 2 contains unique haplotypes obtained from cyt b sequences for all species.

Within P. papatasi, we determined seven unique haplotypes containing seven polymorphic sites with no indels. Total number of substitutions was five (one transition and one transversion). Nucleotide diversity was generally low (θs= 1.409 ± 0.758; θπ= 0.936 ± 0.749) and haplotype diversity was moderate compared to ITS2 sequences (0.516±0.132). The distribution of P. papatasi haplotypes within the Cukurova region is shown in Figure 1B. Haplotype 3 (h3) was the most widespread haplotype within the region followed by Haplotype 1 (h1) sampled from 2 locations. All other haplotypes were unique to their respective sampling locations.

Within P. sergenti, we determined seven unique haplotypes containing six polymorphic sites with one indel. Total number of substitutions was six (four transitions and two transversions). Nucleotide diversity was generally low (θs=1.887 ± 0.996; θπ=1.725 ± 1.199) however haplotype diversity was high 0.850 ± 0.060. The distribution of P. sergenti haplotypes within the Cukurova Region is shown in Figure 1D. Haplotype 2 (h2) was the most widespread haplotype within the region sampled from 4 of the 10 locations.

Among all three species, P. tobbi had the highest number of unique haplotypes (18) with a total of 68 polymorphic sites with no indels. The total number of substitutions was 68 (46 transitions and 23 transversions). Nucleotide diversity was substantially higher than the other two species (θs=17.021 ± 5.518; θπ=9.815 ± 5.137) and haplotype diversity was 0.905 ± 0.038). Figure 1F shows the distribution of P. tobbi haplotypes in the study area. h2 and h5 were the most common and widespread haplotypes while the remaining haplotypes were mostly sampled from one location. Haplotype diversity was highest in the Yemisli (YEM) village with four haplotypes.

Results of separate AMOVA analyses of P. papatasi, P. sergenti and P. tobbi populations are given in Table 3. AMOVA analysis revealed significant genetic structuring between populations for P. papatasi and P. sergenti. In these species, between population genetic variation was higher than within population genetic variation and the associated variance components were significant (P. papatasi, 67.06%, Va = 0.339, p = <0.001; P. sergenti, 66.67%, Va = 0.500, p = <0.001) variation and variance components for between population variation was not significant (P. papatasi, 31.95%, Va=0.347, p=0.122; P. sergenti, –12.46%, Va=-0.106; p=0.653; P. tobbi, 40.27%, Va=0.824, p=0.073). In both P. papatasi and P. sergenti, Fst values were relatively high and significant (P. papatasi, Fst = 0.671; p = 0.003; P. sergenti, Fst = 0.666; p < 0.001) indicating a certain degree of isolation between populations. In P. tobbi however, we did not detect any significant population genetic structuring, as most of the variation was attributed to within population variation (82%) . Genetic variation between populations was low (17%) and the related variance component was not significant (Va = 0.875, p = 0.277). Fst values were also low for populations of P. tobbi (Fst = 0.177; p <0.001) indicating little population isolation.

Results of neutrality tests designed to assess whether nucleotide polymorphism deviated from expectation under neutrality for all species are shown in Table 3. Tajima's D and Fu's Fs values were negative and significant in all three species. The significant negative values of these test statistics might indicate that the Cukurova populations of P. papatasi, P. sergenti and P. tobbi are undergoing processes of demographic expansion.

DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

This study is the first concerning the genetic structure of sand fly populations within Turkey. ITS2 and cyt b gene sequences that are useful markers for intra population level studies were used in order to determine the genetic diversity and structures of P. papatasi, P. sergenti, and P. tobbi populations. In this study, sequences were A-T rich, as expected, which is a common observation for both gene regions (Simmons and Weller 2001, Parvizi and Ready 2006, Depaquit et al. 2002, 2008).

Analyses conducted using the ITS2 gene region revealed no genetic structuring between populations of P. papatasi, P. sergenti and P. tobbi in the Cukurova Region. In contrast, analyses conducted using the cyt b gene region determined the presence of genetic structuring in P. papatasi and P. sergenti populations. However, even though AMOVA analyses returned positive results for P. papatasi and P. sergenti populations, haplotype distributions did not support this conclusion since haplotype distribution was uniform with no apparent geographic clustering. Therefore, we can conclude that even if there is some genetic structuring in P. papatasi and P. sergenti populations according to the cyt b gene region, this is weak at best.

P. papatasi has a widespread distribution between the 20th and 45th latitudes and can be found throughout much of Europe, Asia, and Africa. One would expect such a widespread taxa to have some sort of genetic structuring within its distributional range, but studies dealing with the genetic diversity of P. papatasi have revealed little to no genetic variation within this species distributional range (Esseghir et al. 1997, Parvizi and Ready 2006, Depaquit et al. 2008). Thus, it was not surprising that we found a similar result with P. papatasi populations showing little to no genetic structuring within the Cukurova basin, a small and closed area. Esseghir et al. (1997) determined the presence of 16 haplotypes in 27 P. papatasi specimens collected from 12 countries including India, Iraq, Syria, Cyprus, Spain, Italy, Morocco and Egypt. The papa1 haplotype from the Mediterranean basin was determined to be the ancestral sequence, but nucleotide diversity between haplotypes were low as they were separated from each other by only one to four nucleotide substitutions Researchers explained the lack of genetic structure within the distributional range of P. papatasi by the absence of vicariance in Mediterranean basin and they concluded that the Mediterranean Sea is not an effective barrier to prevent the dispersal of this species (Esseghir et al. 1997). Congruently, Parvizi and Ready (2006) tested for the presence of isolation by distance within P. papatasi populations in Iran. However, they found no support for isolation by distance and gene flow seemed to continue between geographically separated populations. In their study concerning the molecular homogeneity of different geographical populations of P. papatasi, Depaquit et al. (2008) collected P. papatasi specimens from 18 countries and revealed a few number of ITS2 and ND4 (mtDNA) haplotypes shared between populations with no geographic connections. One study where the presence of some genetic diversity was found within P. papatasi was that of Hamarsheh et al. (2007) in which researchers found significant genetic differentiation between distantly separated populations of P. papatasi in relation to mtDNA cyt b sequences. Hamarsheh et al. (2007) stated that the main factor determining the degree of genetic diversity was latitude and not climatic conditions. Researchers also concluded that the determined level of genetic differentiation was not enough to support the existence of a species complex.

The known vector of L. tropica in Southeastern Anatolia, Turkey, P. sergenti, showed no geographical structuring within the Cukurova basin in terms of the distribution of haplotypes obtained from both ITS2 and cyt b gene regions. As one of the most widely distributed sand fly species, P. sergenti has a patchy distribution within its range and sub-populations are significantly different in vector capacities (Depaquit et al. 1998). It has also been pointed out that the distribution of the species is wider than epidemics of leishmaniasis and that this taxon should be treated as a species complex. Depaquit et al. (2000, 2002) revealed P. sergenti populations to be separated in two major lineages pertaining to northeast and south and west populations according to ITS2 sequence variation. They also suggested that the northeast branch is the ancestral clade showing no or very little between population variations, although there are marked differences between populations in terms of ecology, host preference and potential vector capacity. In addition, ITS2 sequences of three populations sampled from Turkey (one population from Adana and two populations from Şanlıurfa) were totally identical. Dvorak et al. (2006) using laboratory colonies (Turkey and Israel) of P. sergenti investigated the two clades in the above study using molecular (RAPD and ITS2 sequences), morphometric (wing shape and size) and cross-mating methods. They found variation between sub groups of Israeli colonies and significant wing deformations between Israeli and Turkish colonies. Also, F1 progeny obtained from the cross-mating experiments formed a distinct group with an intermediate position between the Turkish and Israeli groups, indicating that these two populations, although geographically separated, have not reached reproductive isolation. Yahia et al. (2004) collected populations from different altitudes in Morocco but could not find any significant difference between haplotype frequencies in relation to altitude. However, Baron et al. (2008) detected high diversity within Spanish populations of P. sergenti. In a similar study, Moin-Vaziri et al. (2007) showed that the three different monotypes of P. sergenti within Iran showed no significant genetic differentiation.

Among the three species evaluated in this study, P. tobbi showed the highest number of polymorphic sites and nucleotide diversity. Although haplotype diversity was high in both gene regions, haplotypes showed no geographical structuring within the Cukurova basin. The findings showing P. tobbi to be the vector of L. infantum in the study area have increased the importance of this species (Svobodova et al. 2009). The antropophilic property of the species may stem from the fact that due to the socio-economical conditions prevalent within the region, humans live in close proximity to animals and animal shelters.

Genetic researches conducted on the Larroussius subgenus have usually concentrated on phylogenetic, identification of species complexes or phylogeographic assessments of taxa. Esseghir et al. (1997, 2000) recorded low between-population variation for two Larroussius species, P. perniciosus and P. perfiliewi. West Mediterranean populations of P. perniciosus and P. tobbi were similar to each other both morphologically and genetically, and their populations sampled from Tunisia, Malta, and Italy also showed low haplotype diversity. On the other hand, Parvizi and Assmar (2007) used the nuclear elongation factor – 1α” (EF-1α) and detected seven haplotypes from eight specimens of P. tobbi from Iran, thereby indicating that the diversity was quite high despite the narrow distribution of the species. This study supports our findings of high haplotype diversity of P. tobbi in The Cukurova region.

Statistically significant and negative Tajima's D and Fu's Fs values suggest an excess of rare alleles, population expansion (demographic instability), or purifying selection at the sequenced loci (Nei and Kumar 2000, Paupy 2008). In this study, statistics showed negative values for cyt b gene loci but only Fu's Fs statistics were significant indicating that population expansion more than selection might be responsible for deviations from neutrality, since Fu's Fs value is highly sensitive to range expansions, which lead to high negative values (Fu 1997, Ramos-Onsins and Rozas 2002). Simsek et al. (2007) has claimed that the Taurus and Amanos mountains surrounding the Cukurova basin do not act as barriers limiting the dispersal of sand fly populations. Therefore, our results suggest that vector species within the Cukurova basin are increasing their distributional range. This will undoubtably have important consequences for the spread of vector borne diseases to neighboring areas. Accordingly future studies will be conducted using a larger number of loci and individuals and will focus on the whole of Turkey together with neighboring biogeographic regions. This will enable us to extract not only more detailed and reliable results but will also help us frame our results on a more global scale.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

This work was funded by the Scientific and Technological Research Council of Turkey (TBAG 105 T 205) and Hacettepe University Scientific Researches Unit (05.01.601.005). This study is a part of a Ph.D. thesis submitted to Hacettepe University. We deeply thank Prof. Dr. Cihan Oner, Assoc. Prof. Hatice Mergen, Hasan Unal, MsC, and Dr. Cagatay Karaaslan for helping us during the molecular analyses and for opening their laboratory to us.

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  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices
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Appendices

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
  9. Appendices

Appendix 1

Haplotypes obtained from ITS2 gene region.

A: Ph. papatasi

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B: Ph. sergenti

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C: Ph. tobbi

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Appendix 2

Haplotypes obtained from cyt b gene region.

A: Ph. papatasi

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B: Ph. sergenti

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C: Ph. tobbi

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